Rapid and sensitive Xanthomonas citri detection by LAMP combined with naked eye evaluation methods.

RIGANO L.A.; MARANO M.R.; CASTAGNARO A.P.; DO AMARAL A.M.; VOJNOV A.A.

Tucumán

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Xanthomonas citri pv. citri is the bacterial pathogen that causes the canker disease in citrus plants worldwide. The early and accurate identification of this bacterium in citrus is critical for the protection of this crop industry. Detection methods based on antibodies or PCR has been developed previously, but this approaches are time consuming, laborious and requires expensive laboratory equipment. Loop mediated isothermal amplification (LAMP), a novel isothermal DNA amplification technique, is very sensitive, specific, fast and does not require specialized laboratory equipment. A loop mediated isothermal amplification assay for the detection of Xanthomonas citri pv. citri (Xac-LAMP) was developed and evaluated. The assay was optimized for the amplification of a portion of the pthA gene, a pathogenicity determinant of Xac. A typical ladder like pattern on gel electrophoresis was observed in all positive samples. Amplification products were detected by visual inspection using SybrGreen and using a lateral flow dipstick, eliminating the need of time consuming gel electrophoresis. The sensibility and specificity of the assay were evaluated in different conditions. Not cross reaction was observed with pure DNA of other phytopathogenic bacteria. The Xac-LAMP assay is a simple, fast, sensitive and specific alternative for the accurate detection of Xanthomonas citri pv. citri.